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The Construction, Expression and Purification of Recombinat Human Cardiac Troponin I Protein

Author: SunMuNan
Tutor: TanYan；DuanXiuMei
School: Jilin University
Course: Medical Bioengineering
Keywords: human cardiac troponinⅠ eukaryonization expression system Ni-NTA affinity chromatography
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 117
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Abstract

The human cardiac troponin (hcTn) is a regulatory protein related with cardiac contractility variability, hcTn exists in both cardiac and skeletal muscle as a non-covalently-bound complex of three subunits,the isoforms troponin C,the calcium-binding subunit,troponin I,the inhibitory subunit,and troponin T,which locates the troponin complex on tropomyosin. cTnI encodes 209 amino acid. It’s about 29kD, and its isoelectric point is 9.87.Because of the low molecular weight and cTnI is sensitive to myocardial damage, cTnI releases from cytoplasm firstly as the cardiac muscle is damaged. cTnI will have positive results after micro myocardial injury.cTnI and cTnT complex release sustained and slow from myocardial cells duration up 7 to 10 days. Since early in plasma and lasting longer time than others in serum of cTnI, it appear ideally suited for the early diagnosis of myocardial injury.Troponin I (Tnl) is the inhibitory protein of the troponin-tropomyosin complex. It exists in three isoforms, one for slow-twitch skeletal muscle, one for fast-twitch skeletal muscle, and one for cardiac muscle. These three isoforms of TnI are encoded by three distinct genes. The amino acid sequences of the two skeletal and one cardiac-TnI forms (sTnI and cTnI, respectively) show 40% dissimilarity. Furthermore, the human cTnI has 31 additional amino acid residues on its N-terminal that are not present on the skeletal forms, making this protein a promising analyze for indicating cardiac specificity. cTnI have no cross-reactivity with the skeletal muscle forms. Elevations of cTnI are highly specific for myocardial injury. As traditional methods for isolation of cTnI from human cardiac tissue are tedious and result in relatively low yield of pure material, prokaryotic fusion expression can offer an almost unlimited supply of material without the additional disadvantage of variable human tissue availability. It’s a really complex process to separate and purify cTnI from human myocardium, and genetic engineering technique provides a efficient pathway to solve this problem.The recombination expression system of foreign gene can be divieded into two kinds-eukaryonization and prokaryotic system. Escherichia coli has a clear genetic background, so it has been a first choice to express forgein gene.prokaryotic expression is a method that insert a proper vector into escherichia coli with the purpose of expressing target proteins efficiently which applied in protein purification、location、function analysis and other aspects.The advantage of recombinant protein expressed in Escherichia coli is easy to grow and control, Material for bacterial culture is not expensive, and can choose a variety of E. coli strains and their match with the various features of the plasmid.To purify recombinant protein,6×His tag is constructed to separate the target protein by Ni-NTA affinity chromatography which takes advantage of the affinity between Ni2+ and His.In this article, we mainly resolve the following respects:1.The construction of cTnI prokaryotic expression vectorIn this study, the genes of cardiac troponin I were cloned into pQE30 vector to construct fusion plasmids pQE30-TnI, which were expressed with high efficiency in E.coli M15.2. The expression of Recombinant cTnI in E.coli with IPTG induction.We explored the proper concentration and time for IPTG induction, and finally obtained high efficient expressed recombinant protein which formed mostly inclusion body. So the low concentration of urea was used to wash precipitation.3.By Ni-NTA affinity chromatographyRecombinant protein which contains 6×His was bound with Ni2+, and different concentrations of imidazole which acted as a elution help us gained high purified hcTnI proteins.4.Western Blot analyzitionThe immunological activity of the expressed hcTnI was analyzed by Western Blot after SDS-PAGE, and the result showed that the recombinant protein was characterized by high-specific antigenicity.The natural cTnI could be degradated by proteolytic enzymes even if the recombinant protein existed in the form of inclusion body.TnI forms oligomer under high concentration which is also existed in 8M urea, so it could degradate even under conditions involving protease inhibitor, needing a low temperature situation and addition of enzyme during experiment.In this study, the genes of cardiac troponin I were cloned into pQE30 vector to construct the prokaryotic expression vector pQE30-hcTnI successfully which were expressed with high efficiency in E.coli M15 after inducing with IPTG. cTnI protein was purified by Ni-NTA affinity chromatography and obtained high purity. The immunological activity of the expressed hcTnI was analyzed by Western Blot. This will provide theoretical basis for preparation of high-specific monoclonal antibody and biological function of hcTnI.

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